FIZ1 is expressed during photoreceptor maturation, and synergizes with NRL and CRX at rod-specific promoters in vitro

Abstract

FIZ1 (Flt-3 Interacting Zinc-finger) interacts and co-purifies with the rod-specific transcription factor NRL (Neural Retina Leucine zipper). We hypothesize that FIZ1 is part of an interface between cell-specific factors, like NRL, and more ubiquitous regulatory networks that vary the absolute expression levels of some rod-specific genes (i.e. Rhodopsin). As part of an ongoing exploration of FIZ1's role in neural retina, in vivo, we have taken the first look at FIZ1 expression in the developing mouse retina during the retinal maturation period. Using the normal C57/B6 mouse as a model, multiple approaches were used including: immunoblotting, immunohistochemistry, and quantitative real-time PCR. Functional implications of FIZ1/NRL interaction, on NRL-and CRX-mediated activation of the Rhodopsin (Rho) and cGMPphosphodiesterase β-subunit gene (PDE6B) promoters, were examined by co-transfection assays. Immunoblot analysis revealed that FIZ1 protein levels were lowest in immature mouse neural retina (P0). FIZ1 concentration increased at least ten-fold as the neural retina matured to the adult state (P21 and later). Immunohistochemical comparison of immature post-natal and mature adult retina revealed increasing FIZ1 protein in photoreceptors, the inner plexiform layer, and the ganglion cell layer. Total retinal Fiz1 mRNA content increased as the neural retina matured. The expected increase in Rho mRNA level was also monitored as a genetic marker of photoreceptor maturation. In transient co-transfection assays of CV1 cells, FIZ1 synergized with NRL to activate transcription from the Rho and PDE6B gene promoters with some differences. In the case of the Rho promoter, FIZ1 synergized when both NRL and CRX were present. With the PDE6B promoter, FIZ1 synergized with NRL alone, and the inclusion of CRX decreased this synergy.Conclusions-These findings support previous evidence that FIZ1 is present in rodphotoreceptors. (Co-immunoprecipitation from nuclear-protein extracts with rod-specific NRL) FIZ1 expression increases in the neural retina during the retinal maturation period. Additionally, in vitro experiments demonstrate that FIZ1 has the potential to significantly increase the NRLmediated activation of photoreceptor-specific promoters. While CRX is not a strong activator of the PDE6B promoter, alone or with NRL, CRX decreased the synergy of NRL with FIZ1.

FIZ1 is part of the regulatory protein complex on active photoreceptor-specific gene promoters in vivo

“…Samples were incubated with 0.1 M glycine solution for 15 minutes and washed with PBS-T (3 × 5 min). Samples were blocked for non-specific protein binding with 5% normal goat serum in PBS-T for 1 hour and incubated with anti-bFIZ1 antibody (1:100, 4°C), a rabbit IgG made to target domain-II-IV of FIZ1 (Proteintech Group Inc., Chicago, IL, USA) that was affinity purified and specific for FIZ1 in immunoblots of mammalian retina [ 11 , 13 ]. After washing with PBS-T (3 × 5 min), samples were incubated with Nanogold-Fab'goat anti-rabbit-IgG nano gold (gold particle size 1.5 nm maximum, 1.4 nm average), 1:50 in PBS-T, 1 hour at 37°C (Nanoprobes, Yaphank, NY).…”

“…While FIZ1 mRNA is found in most human and mouse tissues, FIZ1 protein content is lower in immature postnatal mouse retina and its concentration increases ten-fold (after P-5) as retinal neurons mature to full functionality [ 11 , 12 ]. Immunohistochemistry has revealed increasing FIZ1 concentration in photoreceptors, the inner plexiform layer, and the ganglion cell layer [ 13 ]. Functionally, FIZ1 can synergize with NRL and CRX to increase the activation of the Rho and PDE6B (human rod Phosphodiesterase beta subunit) promoters in vitro [ 13 ].…”

“…Immunohistochemistry has revealed increasing FIZ1 concentration in photoreceptors, the inner plexiform layer, and the ganglion cell layer [ 13 ]. Functionally, FIZ1 can synergize with NRL and CRX to increase the activation of the Rho and PDE6B (human rod Phosphodiesterase beta subunit) promoters in vitro [ 13 ].…”

Regulation of photoreceptor gene expression by Crx-associated transcription factor network

“…Nrl was also reported to interact with Fiz1, a zinc finger protein that complexes with Flt3 receptor tyrosine kinase (Mitton et al, 2003). Fiz1 potentiates Nrl or Crx/Nrl activity on rhodopsin and PDE6B promoters (Mali et al, 2007), further implicating Nrl's involvement in coordinating the intrinsic photoreceptor developmental program with extracellular signaling pathways.…”

Mutation inBmpr1bLeads to Optic Disc Coloboma and Ventral Retinal Gliosis in Mice

“…PDE6B is a marker for rod photoreceptors. 31,32 PDE6B immunostaining demonstrated a disruption and delamination of the photoreceptor layers in the mutant ventral retina compared to the wild types (Figs. 3c, A and E).…”